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J. Agric. Food Chem. 2009, 57, 6334–6341 DOI:10.1021/jf901117z
Differentiation in Fatty Acid Profiles of Pigmented and Nonpigmented Aurantiochytrium Isolated from Hong Kong Mangroves KING-WAI FAN,† YUE JIANG,*,‡ LOK-TANG HO,† AND FENG CHEN*,† †
School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong, and ‡ Department of Biology, Kwong Living Trust Food Safety & Analysis Laboratory and Sino-Forest Applied Research Centre for the Pearl River Delta Environment, Hong Kong Baptist University, Hong Kong
Twenty-five thraustochytrids that belong to the genus Aurantiochytrium were isolated from subtropical mangroves in Hong Kong. Although they have similar morphological and physiological characteristics, they have different colors on a yeast extract-glucose agar plate, which were largely ignored before. On the basis of the differences in their colony color, 25 Aurantiochytrium strains were further classified into pigmented and nonpigmented subgroups and their fatty acid profiles were analyzed and compared. In general, nonpigmented Aurantiochytrium strains were found to contain biomass concentrations and growth yield coefficients statistically higher than pigmented Aurantiochytrium strains (p < 0.01). Among all isolates, a significantly higher content of polyunsaturated fatty acid (PUFA, 123.41-179.64 mg/g) was found in the nonpigmented Aurantiochytrium (p < 0.01), whereas the pigmented strains contained a higher amount of saturated fatty acids. Docosahexaenoic acid (DHA) was identified as the most abundant PUFA in both nonpigmented and pigmented Aurantiochytrium. According to the result of principal component analysis, the contents and composition of saturated fatty acids and PUFAs are the major varieties to distinguish these two Aurantiochytrium groups, especially the contents of C15:0, C13:0, C16:0, C17:0, and DHA. With a rapid growth rate and high DHA yield, the strain from the nonpigmented Aurantiochytrium group was regarded as the ideal candidate for PUFA production. KEYWORDS: Fatty acid; thraustochytrid; Aurantiochytrium; DHA
INTRODUCTION
Long chain polyunsaturated fatty acids (PUFAs) are essential constituents of cell membranes and cell signaling systems (1). Some of them such as docosahexaenoic acid (DHA, ω-3 C22:6,) and arachidonic acid (AA, ω-6 C20:4) are major components of gray matter of brain as well as precursors of docosanoids and eicosanoids, respectively. They are important for the early cognitive and visual development of infants and therefore have now been used as added components in infant formulas in developed countries worldwide (2-4). Recently, it was proven by clinical evidence that PUFAs are able to alleviate symptoms of certain diseases such as coronary heart disease, stroke and rheumatoid arthritis (5). Because of the noticeable importance of PUFAs in human health and nutrition, different means are used to increase the human consumption of PUFAs from different food sources such as direct intake as food additives and nutraceuticals as well as indirect consumption via the enrichment of PUFAs in important species in aquaculture (6). *To whom correspondence should be addressed. (Y.J.) Tel: 8523411-7062. Fax: 852-3411-5995. (F.C.) Tel.: 852-2299-0309. Fax: 8522299-0311.
pubs.acs.org/JAFC
Published on Web 06/17/2009
The major commercial source of PUFAs is currently from marine fatty fish. However, fish and fish oil are reported to contain dioxins, PCBs, heavy metals, and pharmaceutical residues including synthetic estrogens that are a hazard upon longterm exposure (7). Moreover, the seafood has now been identified as a leading source of mercury exposure for humans. As such, the safety of fatty fish and fish oil being used as conventional sources of PUFAs is doubtful. More efforts have therefore been exerted to explore the alternative PUFA sources. The microbial group - thraustochytrid, in particular, has been regarded as the most successful alternative, because some genera are known to contain substantial amounts of lipids, especially long chain polyunsaturated fatty acids, that is, DHA, and can grow well heterotrophically in a stainless-steel fermentor (6, 8, 9). Thraustochytrids are marine osmoheterotrophs with characteristic ectoplasmic net elements for nutrient absorption and are responsible for carbon recycling in marine habitats (10). Although there are six categories of PUFA profiles in thraustochytrids including docosapentaenoic acid (DPA)/DHA, eicosapentaenoic acid (EPA)/DHA, EPA/DPA/DHA, arachidonic acid (AA)/EPA/DHA, linoleic acid (LA)/AA/DPA/DHA, and LA/AA/EPA/DHA (11), the major characteristic of the
© 2009 American Chemical Society
Article
J. Agric. Food Chem., Vol. 57, No. 14, 2009
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Figure 1. Neighbor-joining trees of thraustochytrids and labyrinthulids based on 18S rRNA gene sequence analysis. ( indicates the strain in this study. Bootstrap values (%) were obtained with 1000 replicates and are shown at the nodes. The tree was rooted with Bacillaria paxillifer and Ochromonas danica as outgroup.
commercially important strains reported to date is their higher contents of DHA (12). Mangroves are unique intertidal wetlands largely confined to coastal regions between 30° north and south of the equator and are nursery grounds for organisms ranging from migratory birds and mudskippers to microorganisms prevailing in the water columns of the habitats (13). Mangrove areas were reported to be the most selected sites for the isolation of thraustochytrids owning largely to the heterogeneity of the environments that may exert evolutionary pressure for nurturing thraustochytrids with
rapid growth (8). Hong Kong, located at 22°300 N and 114°100 E, is a rare urban location with its vast mangrove distribution in the south China sea region. Its mangrove distribution is representative of that in the south China sea region (14). In recent field expeditions of several Hong Kong mangrove areas, we found that some Aurantiochytrium strains differed in fatty acid profiles and heterotrophic growth characteristics from those previously isolated strains which contain relatively higher PUFAs contents (15). Moreover, the colony color of the strains exhibit a distinctive reddish orange color (named as pigmented strains in this study)
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Figure 2. Kinetic parameters of growth and glucose consumption of the Aurantiochytrium strains. (A) Maximum biomass concentration Xmax (g/L); (gray bars) nonpigmented Aurantiochytrium spp.; (hatched bars) pigmented Aurantiochytrium spp.; (B) growth yield coefficient based on glucose Yx/glu (g/g). Values are represented as mean ( standard deviation of triplicates and statistically analyzed at a level of p < 0.05.
on glucose-yeast extract agar which is markedly different from the pale creamy color (nonpigmented) of the PUFA-rich strains isolated previously by our group, although they share similar morphological and physiological characteristics (16). Specifically, the pigmented and nonpigmented strains can be further distinguished based on their intracellular carotenoid contents when detected using an HPLC-photodiode array detector. All the pigmented strains contain carotenoid with the content of some strains reaching as high as 100 μg/g of biomass, while the nonpigmented strains do not possess a detectable level of carotenoid (data not shown). In this study, we aimed to investigate and compare the fatty acid profiles and the heterotrophic growth properties of the pigmented and nonpigmented thraustochytrid Aurantiochytrium, a frequently isolated genus from Hong Kong mangrove areas. A randomly selected strain categorized by
morphological and physiological analyses was chosen as a representative for subsequent confirmation using 18s rRNA gene analysis. The output from this research would not only provide comprehensive information on the fatty acid production characteristics of these subtropical thrasutochytrids but also facilitate further investigation of thraustochytrid for its potential application in the production of other useful products, that is, pigments. MATERIALS AND METHODS
Heterotrophic Growth. The isolation of thraustochytrid was conducted in different mangrove areas in the Deep Bay and Sai Kung districts during the summer of 2007 according to the isolation method described by Fan et al. (16). Three mangrove sites, namely, Luk Keng (LK), Ting Kok (TK), Sai Keng (SK) in Sai Kung and the largest mangrove stands in the Hong Kong - Mai Po Nature Reserve (MP) in the Deep Bay region were
e
NP NP NP NP NP NP NP NP NP NP NP NP P P P P P P P P P P P P P
30 30 30 30 30 30 25 25 15-18 15-18 8-10 8-10 30 25 25 25 25 15-18 15-18 15-18 15-18 15-18 15-18 15-18 8-10
14:0
5.66 ( 0.37 5.61 ( 0.19 4.54 ( 0.45 5.60 ( 0.51 4.74 ( 0.44 4.93 ( 0.26 4.92 ( 0.17 4.67 ( 0.18 4.93 ( 0.36 4.54 ( 0.17 4.43 ( 0.21 4.60 ( 0.35 6.09 ( 0.70 8.06 ( 0.67 14.93 ( 2.10 11.81 ( 2.16 5.19 ( 0.04 5.80 ( 0.93 15.64 ( 0.81 6.95 ( 1.43 10.33 ( 1.63 15.32 ( 2.27 11.08 ( 1.20 14.52 ( 0.72 15.60 ( 1.41
13:0
0.04 ( 0.01 0.10 ( 0.03 0.09 ( 0.01 0.16 ( 0.02 0.08 ( 0.01 0.10 ( 0.00 0.09 ( 0.00 0.08 ( 0.00 0.08 ( 0.00 0.08 ( 0.00 0.10 ( 0.01 0.12 ( 0.00 3.96 ( 0.76 3.60 ( 0.38 2.16 ( 0.11 2.65 ( 0.34 2.39 ( 0.01 1.47 ( 0.25 1.16 ( 0.29 1.98 ( 0.13 2.04 ( 0.27 1.78 ( 0.23 2.21 ( 0.37 1.46 ( 0.08 1.79 ( 0.34
4.22 ( 0.23 4.11 ( 0.25 4.78 ( 0.33 5.89 ( 1.00 4.90 ( 0.63 4.92 ( 0.13 4.69 ( 0.12 4.14 ( 0.13 4.16 ( 0.37 3.93 ( 0.18 5.69 ( 0.38 5.86 ( 0.01 40.20 ( 1.17 39.18 ( 1.81 27.82 ( 3.85 34.16 ( 4.11 32.87 ( 0.20 30.82 ( 2.83 21.98 ( 4.69 31.72 ( 2.74 32.33 ( 4.87 26.20 ( 3.52 32.80 ( 4.50 23.52 ( 3.55 26.33 ( 5.18
15:0 57.50 ( 0.56 52.88 ( 0.89 50.77 ( 2.13 49.76 ( 0.51 53.75 ( 0.31 52.60 ( 0.39 52.92 ( 0.17 53.06 ( 0.62 54.06 ( 0.76 55.84 ( 1.08 48.10 ( 0.94 50.74 ( 0.20 15.67 ( 1.56 15.90 ( 0.49 33.24 ( 3.53 25.81 ( 3.57 18.86 ( 0.11 22.01 ( 0.57 37.16 ( 2.34 24.36 ( 1.81 25.55 ( 3.44 32.98 ( 3.03 25.99 ( 4.16 31.84 ( 2.20 31.38 ( 4.47
16:0 0.89 ( 0.18 0.83 ( 0.07 0.96 ( 0.08 1.21 ( 0.17 0.97 ( 0.12 1.02 ( 0.09 1.06 ( 0.04 0.85 ( 0.04 0.91 ( 0.09 0.79 ( 0.02 1.33 ( 0.11 1.38 ( 0.00 8.01 ( 0.18 8.02 ( 0.52 4.67 ( 0.48 6.02 ( 0.97 7.79 ( 0.04 6.47 ( 0.12 3.72 ( 0.62 5.84 ( 0.14 5.40 ( 0.67 5.07 ( 0.43 5.79 ( 0.37 3.85 ( 0.21 3.97 ( 0.82
17:0 1.52 ( 0.20 1.37 ( 0.13 1.21 ( 0.12 1.18 ( 0.03 1.29 ( 0.00 1.29 ( 0.00 1.30 ( 0.04 1.26 ( 0.01 1.38 ( 0.01 1.39 ( 0.03 1.18 ( 0.04 1.10 ( 0.02 0.46 ( 0.11 0.38 ( 0.02 0.84 ( 0.02 0.67 ( 0.05 0.84 ( 0.04 0.55 ( 0.08 0.93 ( 0.10 0.69 ( 0.03 0.62 ( 0.04 0.79 ( 0.08 0.60 ( 0.09 0.75 ( 0.05 0.69 ( 0.06
18:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.24 ( 0.02 0.15 ( 0.01 0.20 ( 0.04 0.16 ( 0.03 0.25 ( 0.04 0.15 ( 0.02 0.29 ( 0.07 0.23 ( 0.04 0.18 ( 0.00 0.24 ( 0.02 0.17 ( 0.01 0.19 ( 0.03 0.29 ( 0.03
16:1 n-7 0.25 ( 0.05 0.29 ( 0.03 0.30 ( 0.02 0.52 ( 0.05 0.33 ( 0.06 0.52 ( 0.06 0.59 ( 0.07 0.33 ( 0.04 0.41 ( 0.01 0.26 ( 0.01 0.58 ( 0.08 0.32 ( 0.07 0.40 ( 0.12 0.11 ( 0.00 0.25 ( 0.02 0.31 ( 0.06 0.68 ( 0.07 0.10 ( 0.02 0.22 ( 0.03 0.31 ( 0.04 0.12 ( 0.01 0.51 ( 0.00 0.18 ( 0.02 0.33 ( 0.03 0.27 ( 0.05
18:1 n-9
monounsaturated fatty acid
0.70 ( 0.11 0.78 ( 0.12 0.78 ( 0.07 0.94 ( 0.16 0.74 ( 0.12 1.15 ( 0.17 1.51 ( 0.10 0.73 ( 0.03 0.82 ( 0.10 0.72 ( 0.00 1.31 ( 0.00 1.04 ( 0.28 1.02 ( 0.12 0.84 ( 0.08 0.59 ( 0.07 0.71 ( 0.04 1.64 ( 0.02 0.78 ( 0.20 0.47 ( 0.00 0.67 ( 0.01 0.75 ( 0.06 0.63 ( 0.02 0.69 ( 0.04 0.78 ( 0.02 0.75 ( 0.14
AA 0.42 ( 0.03 0.45 ( 0.03 0.48 ( 0.02 0.47 ( 0.04 0.42 ( 0.02 0.41 ( 0.01 0.45 ( 0.01 0.45 ( 0.02 0.46 ( 0.04 0.44 ( 0.01 0.54 ( 0.01 0.49 ( 0.01 0.36 ( 0.03 0.37 ( 0.03 0.21 ( 0.03 0.25 ( 0.02 0.53 ( 0.00 0.46 ( 0.07 0.28 ( 0.01 0.44 ( 0.08 0.36 ( 0.02 0.23 ( 0.01 0.34 ( 0.02 0.31 ( 0.04 0.29 ( 0.03
EPA 4.54 ( 0.58 4.96 ( 0.05 5.11 ( 1.00 4.61 ( 0.44 4.89 ( 0.73 3.97 ( 0.46 3.56 ( 0.24 5.07 ( 0.29 5.00 ( 0.45 5.16 ( 0.16 5.02 ( 0.06 5.20 ( 0.43 5.56 ( 0.21 5.46 ( 0.35 3.45 ( 0.16 3.87 ( 0.09 7.00 ( 0.08 7.92 ( 0.75 4.26 ( 0.59 6.88 ( 0.50 5.47 ( 0.21 3.68 ( 0.40 5.06 ( 0.12 5.32 ( 0.41 4.45 ( 0.18
DPA
polyunsaturated fatty acid
23.53 ( 1.26 27.66 ( 0.48 30.01 ( 2.32 28.79 ( 0.06 27.18 ( 1.08 28.03 ( 0.70 27.76 ( 0.24 28.64 ( 0.48 27.03 ( 0.03 26.12 ( 0.90 30.78 ( 0.43 28.32 ( 0.19 16.08 ( 0.28 16.29 ( 1.26 10.16 ( 0.88 11.73 ( 0.24 18.29 ( 0.21 21.40 ( 1.86 12.25 ( 1.49 18.34 ( 1.62 14.79 ( 0.45 11.04 ( 0.70 13.36 ( 0.03 14.88 ( 0.38 12.22 ( 0.37
DHA 41.85 ( 3.72 37.83 ( 3.06 49.03 ( 1.72 44.89 ( 4.49 50.95 ( 4.76 51.43 ( 1.68 48.03 ( 0.19 50.01 ( 2.53 50.11 ( 0.23 44.34 ( 3.26 45.80 ( 1.46 40.18 ( 4.89 22.80 ( 1.84 25.39 ( 3.52 47.74 ( 5.27 37.75 ( 3.00 18.58 ( 0.05 23.97 ( 5.02 47.53 ( 3.66 22.83 ( 5.62 45.52 ( 2.11 38.17 ( 3.94 34.29 ( 0.87 40.38 ( 4.92 44.02 ( 3.53
TFAd 30.36 ( 1.52 35.20 ( 0.78 37.78 ( 3.10 36.50 ( 0.06 34.56 ( 1.50 35.46 ( 0.79 35.61 ( 0.17 36.18 ( 0.24 34.76 ( 0.17 33.63 ( 1.09 39.79 ( 0.62 36.62 ( 0.03 24.82 ( 0.65 24.28 ( 1.80 15.56 ( 1.18 17.97 ( 0.31 30.55 ( 0.48 31.81 ( 3.10 18.38 ( 2.17 27.77 ( 2.33 22.60 ( 0.49 17.05 ( 1.06 20.68 ( 0.27 22.75 ( 0.80 19.17 ( 0.33
unsatde
295.09 ( 15.54 249.05 ( 14.12 310.66 ( 9.14 291.58 ( 11.32 338.49 ( 18.21 340.43 ( 11.91 319.37 ( 8.75 324.42 ( 12.31 333.09 ( 6.85 298.83 ( 9.11 284.46 ( 5.20 260.18 ( 8.21 175.24 ( 7.69 195.37 ( 9.12 408.38 ( 19.84 314.18 ( 13.48 134.02 ( 3.11 166.64 ( 8.75 392.99 ( 16.57 168.28 ( 9.38 357.08 ( 14.51 322.13 ( 11.19 275.58 ( 8.54 317.03 ( 18.15 361.53 ( 14.13
SFAf (mg/g)
1.05 ( 0.06 1.10 ( 0.07 1.47 ( 0.07 2.33 ( 0.11 1.68 ( 0.10 2.67 ( 0.12 2.83 ( 0.15 1.65 ( 0.09 2.05 ( 0.10 1.15 ( 0.06 2.66 ( 0.12 1.29 ( 0.05 1.46 ( 0.06 0.66 ( 0.03 2.15 ( 0.10 1.77 ( 0.08 1.73 ( 0.07 0.60 ( 0.01 2.42 ( 0.11 1.23 ( 0.02 1.37 ( 0.05 2.86 ( 0.12 1.20 ( 0.05 2.10 ( 0.10 2.47 ( 0.12
MUFAf (mg/g)
123.41 ( 17.95 129.25 ( 12.84 179.64 ( 9.35 157.32 ( 16.48 171.01 ( 24.51 173.87 ( 10.92 160.93 ( 2.30 175.68 ( 10.07 168.01 ( 0.63 144.57 ( 5.84 173.54 ( 3.08 141.62 ( 15.94 52.76 ( 2.72 58.53 ( 3.85 69.02 ( 2.24 63.32 ( 3.65 51.78 ( 0.46 73.06 ( 8.48 82.31 ( 3.51 60.02 ( 9.82 98.12 ( 7.11 59.57 ( 1.96 67.32 ( 0.95 86.77 ( 13.70 78.67 ( 7.99
PUFA f (mg/g)
98.71 ( 14.02 104.71 ( 10.26 146.91 ( 6.21 129.19 ( 12.64 138.76 ( 18.44 144.23 ( 8.28 133.34 ( 1.69 143.26 ( 9.68 135.43 ( 0.75 115.67 ( 4.53 140.93 ( 2.53 113.77 ( 13.09 36.63 ( 2.32 41.14 ( 2.55 48.29 ( 1.17 44.26 ( 2.61 33.98 ( 0.29 50.82 ( 6.28 57.95 ( 2.62 41.41 ( 6.62 67.35 ( 5.18 41.99 ( 1.69 45.82 ( 1.04 60.19 ( 8.85 53.88 ( 5.96
DHAg (mg/g)
a Data are expressed as mean ( SD of three replicates and statistically analyzed at a level of p
